Clinical epidemiology and high genetic diversity amongst Cryptococcus spp. isolates infecting people living with HIV in Kinshasa, Democratic Republic of Congo.

Neuromeningeal cryptococcosis (NMC) is a life-threatening opportunistic infection in advanced HIV disease patients (AHDP). It is caused by Cryptococcus spp. complexes and mainly occurs in sub-Saharan Africa. In this study, we performed molecular characterization and antifungal susceptibility profiling of Cryptococcus isolates from AHDP in Kinshasa (DRC). Additionally, we investigated a possible association between NMC severity factors and the Cryptococcus neoformans (Cn) multilocus sequence typing (MLST) profiles. We characterized the isolates using PCR serotyping, MALDI-TOF MS, internal transcribed spacer (ITS) sequencing, and MLST. Susceptibility testing for the major antifungal drugs was performed according to the EUCAST guidelines. Parameters associated with NMC severity, such as hypoglycorrhachia (< 50 mg/dL), increased cerebral spinal fluid opening pressure (> 30 cm H2O), and poor therapeutic outcome were compared with the Cn MLST sequences type (ST). Twenty-three out of 29 Cryptococcus isolates were identified as serotype A using PCR serotyping (79.3%; 95% IC: 65.5-93.1), while six (20.7%; 95% IC: 6.9-34.5) were not serotypable. The 29 isolates were identified by ITS sequencing as follows: Cryptococcus neoformans (23/29, 79.3%), Cutaneotrichosporon curvatus (previously called Cryptococcus curvatus) (5/29, 17.2%), and Papiliotrema laurentii (Cryptococcus laurentii) (1/29, 3.5%). Using the ISHAM MLST scheme, all Cn isolates were identified as molecular type VNI. These comprised seven different STs: ST93 (n = 15), ST5 (n = 2), ST53 (n = 1), ST31 (n = 1), ST4 (n = 1), ST69 (n = 1), and one novel ST that has not yet been reported from other parts of the world and was subsequently assigned as ST659 (n = 2). Of the included strains, only Papiliotrema laurentii was resistant to amphoterin B (1/29, 3.5%), 6.8% (2/29) were resistant to 5-flucytosine (the single Papiliotrema laurentii strain and one Cryptococcus neoformans isolate), and 13.8% (4/29) to fluconazole, including two of five (40%) Cutaneotrichosporon curvatus and two of 23 (8.7%) C. neoformans strains. We found a significative association between poor therapeutic outcome and a non-ST93 sequence type of causative strains (these concerned the less common sequence types: ST53, ST31, ST5, ST4, ST659, and ST69) (87.5% versus 40%, p = 0.02). Molecular analysis of Cryptococcus spp. isolates showed a wide species diversity and genetic heterogenicity of Cn within the VNI molecular type. Furthermore, it is worrying that among included strains we found resistances to several of the commonly used antifungals.

Community led: celebrating wellness with a smile.

Dental caries is the most common chronic childhood disease in Canada and creates a significant burden on both human and financial costs. In Canada, the annual cost of dental day surgery for children is $21.2 million. The objective of this study was to explore and address the strengths and barriers related to the provision of oral health services in an Indigenous community in northern Saskatchewan. This community-based participatory research project focused on developing authentic relationships with the community. This research is novel because it is community-led and from the perspective of Indigenous people. Descriptive statistics were undertaken to describe the 38 participants. Semi-structured interviews were conducted with elders, healthcare providers, teachers and parents/guardians of elementary school-aged children; and inductive, thematic analysis was undertaken with the qualitative data. The most commonly identified themes included: community resilience, the need to improve oral health literacy and skills and the mitigation of barriers to access care. The research process included co-creating tools with the community that built upon strengths, creating opportunities for change, generated solutions and transforming the health system the community accessed.

Diagnosis and Treatment Approaches to a "Gummy Smile".

Excessive gingival display or "gummy smile" is a growing concern to dental patients and often considered detrimental to an esthetic smile. Gingival display of more than 4 mm of gingiva is considered by many to be unattractive. The cause of the gummy smile can be multifactorial and must be accurately diagnosed to render appropriate treatment. Factors that contribute to the gummy smile include altered passive eruption, lip length, lip hypermobility, incisal wear/crown length, and vertical maxillary excess and gingival hyperplasia. The purpose of this article is to review the etiology, diagnosis, and surgical approaches in treating the gummy smile.

Endotracheal Tube Obstruction via Turbinectomy During Nasal Intubation.

The purpose of this article is to describe a case of an accidental turbinectomy during nasal intubation for an elective oral and maxillofacial surgical case that was confirmed after extubation. While there are several reported cases, this still tends to be an overall rare complication in the field of anesthesia. This article highlights the complications encountered due to turbinectomy while also identifying causes, signs, and methods to prevent it.

Group A streptococcal meningitis: emm type distribution and theoretical vaccine coverage in children.

As group A Streptococcus (GAS) meningitis is seldom reported in children, emm-type distribution data are scare. We report eight cases of GAS meningitis in Belgium (2008-2013) and compare molecular characteristics of our strains with a further 55 cases previously reported with their corresponding emm-types. emm1 type was the most frequent (24%) followed by emm6 (11%), emm12 (11%) and emm3 (6%). Together these four emm-types accounted for 52% of the cases, while the rest of the cases are due to 24 different emm-types. These 28 emm-types associated with GAS meningitis belonged to 16 different emm-clusters suggesting that there is no propensity for particular emm-types or emm-cluster to cause meningitis. Theoretical coverage of the 30-valent vaccine candidate would be 77.8% (49/63 isolates) among children with GAS meningitis.

Streptococcus pyogenes strains in Sao Paulo, Brazil: molecular characterization as a basis for StreptInCor coverage capacity analysis.

BACKGROUND: Several human diseases are caused by Streptococcus pyogenes, ranging from common infections to autoimmunity. Characterization of the most prevalent strains worldwide is a useful tool for evaluating the coverage capacity of vaccines under development. In this study, a collection of S. pyogenes strains from Sao Paulo, Brazil, was analyzed to describe the diversity of strains and assess the vaccine coverage capacity of StreptInCor. METHODS: Molecular epidemiology of S. pyogenes strains was performed by emm-genotyping the 229 isolates from different clinical sites, and PCR was used for superantigen profile analysis. The emm-pattern and tissue tropism for these M types were also predicted and compared based on the emm-cluster classification. RESULTS: The strains were fit into 12 different emm-clusters, revealing a diverse phylogenetic origin and, consequently, different mechanisms of infection and escape of the host immune system. Forty-eight emm-types were distinguished in 229 samples, and the 10 most frequently observed types accounted for 69 % of all isolates, indicating a diverse profile of circulating strains comparable to other countries under development. A similar proportion of E and A-C emm-patterns were observed, whereas pattern D was less frequent, indicating that the strains of this collection primarily had a tissue tropism for the throat. In silico analysis of the coverage capacity of StreptInCor, an M protein-conserved regionally based vaccine candidate developed by our group, had a range of 94.5 % to 59.7 %, with a mean of 71.0 % identity between the vaccine antigen and the predicted amino acid sequence of the emm-types included here. CONCLUSIONS: This is the first report of S. pyogenes strain characterization in Sao Paulo, one of the largest cities in the world; thus, the strain panel described here is a representative sample for vaccine coverage capacity analysis. Our results enabled evaluation of StreptInCor candidate vaccine coverage capacity against diverse M-types, indicating that the vaccine candidate likely would induce protection against the diverse strains worldwide.

Post-infectious group A streptococcal autoimmune syndromes and the heart.

There is a pressing need to reduce the high global disease burden of rheumatic heart disease (RHD) and its harbinger, acute rheumatic fever (ARF). ARF is a classical example of an autoimmune syndrome and is of particular immunological interest because it follows a known antecedent infection with group A streptococcus (GAS). However, the poorly understood immunopathology of these post-infectious diseases means that, compared to much progress in other immune-mediated diseases, we still lack useful biomarkers, new therapies or an effective vaccine in ARF and RHD. Here, we summarise recent literature on the complex interaction between GAS and the human host that culminates in ARF and the subsequent development of RHD. We contrast ARF with other post-infectious streptococcal immune syndromes - post-streptococcal glomerulonephritis (PSGN) and the still controversial paediatric autoimmune neuropsychiatric disorders associated with streptococcal infections (PANDAS), in order to highlight the potential significance of variations in the host immune response to GAS. We discuss a model for the pathogenesis of ARF and RHD in terms of current immunological concepts and the potential for application of in depth "omics" technologies to these ancient scourges.

Severe group A streptococcal infections in a paediatric intensive care unit.

AIM: To describe the clinical presentation, management and outcomes for children with invasive group A streptococcal (GAS) infection in a paediatric intensive care unit (PICU). METHODS: We reviewed the clinical and laboratory records of patients admitted to a PICU in Melbourne with invasive GAS infection from April 2010 to April 2013. Outcomes recorded included survival, organ failure, need for extracorporeal support, renal replacement therapy and prolonged neuromuscular weakness. RESULTS: Twelve cases of invasive GAS infection were identified. The most common clinical presentations were pneumonia (n=5), bacteraemia with no septic focus (n=4) and septic arthritis (n=3). Necrotising fasciitis occurred in one patient and another patient presented with ischaemic lower limbs requiring amputation. Of the eight isolates with available emm typing results, the most common emm type was emm1 (n=4) followed by emm4, 12 and 22. Nine patients had multi-organ failure. Ten patients required mechanical ventilation for a median duration of 8 days. Nine patients required inotropic and/or vasopressor support and four patients extracorporeal membrane oxygenation support. Eleven patients survived. A prolonged period of neuromuscular weakness following the initial severe illness was common (n=5), but most children returned to normal or near normal neurological function. CONCLUSIONS: Invasive GAS disease in children may cause severe multi-organ failure with resultant prolonged intensive care stay and significant morbidity. However, a high rate of survival and return to normal functioning may be achieved with multi-system intensive care support and multi-disciplinary rehabilitation.

Group A streptococcus meningitis in children.

OBJECTIVE: To characterize the epidemiologic burden and the molecular determinants of group A streptococcal (GAS) meningitis among the pediatric population of the state of Paraná, Brazil. METHODS: Clinical and epidemiologic data were gathered by a compulsory notification system during the period 2003 to 2011. Bacterial identification, antibiotic resistance profile, emm-typing, pulsed-field gel electrophoresis typing and virulence profile were analyzed by a central reference laboratory. A review of published pediatric cases of GAS meningitis from the last 45 years was undertaken and compared with the Brazilian series. RESULTS: The incidence of GAS meningitis among the pediatric population was 0.06 cases per 100,000 children per year and was associated with a case fatality rate of 43%. Neonatal age and the presence of an associated toxic shock syndrome were identified as risk factors for death. A distant focus of infection was present in more than half of the patients in the literature and in 36% in the Brazilian case series. A high diversity of emm-types was associated with GAS meningitis in Brazil. No single virulence determinant could be associated with death. CONCLUSIONS: GAS meningitis is associated with high mortality and with a high diversity of GAS emm-types and virulence determinants in Brazil.

Molecular markers for the study of streptococcal epidemiology.

Diseases caused by Streptococcus pyogenes (Group A streptococcus, GAS) range from superficial infections such as pharyngitis and impetigo to potentially fatal rheumatic heart disease and invasive disease. Studies spanning emm-typing surveillance to population genomics are providing new insights into the epidemiology, pathogenesis, and biology of this organism. Such studies have demonstrated the differences that exist in the epidemiology of streptococcal disease between developing and developed nations. In developing nations, where streptococcal disease is endemic, the diversity of GAS emm-types circulating is much greater than that found in developed nations. An association between emm-type and disease, as observed in developed countries is also lacking. Intriguingly, comparative genetic studies suggest that emm-type is not always a good predictor of the evolutionary relatedness of geographically distant isolates. A view of GAS as a highly dynamic organism, in possession of a core set of virulence genes that contribute to host niche specialization and common pathogenic processes, augmented by accessory genes that change the relative virulence of specific lineages is emerging. Our inability to definitively identify genetic factors that contribute to specific disease outcome underscores the complex nature of streptococcal diseases.

Group a streptococcus antibiotic resistance in southern Brazil: a 17-year surveillance study.

BACKGROUND: Scarce data are available about the antimicrobial resistance of Group A Streptococcus in South America. METHODS: This study evaluated the antimicrobial susceptibility profile of 1,112 isolates of Group A Streptococcus during the period from 1993 to 2009 in Curitiba city, Brazil. Macrolide-resistant isolates were characterized by emm typing and pulsed-field gel electrophoresis. RESULTS: All isolates were susceptible to penicillin, vancomycin, and tigecycline. On the contrary, 18.6% of the isolates were resistant to tetracycline, presenting a minimum inhibitory concentration (MIC)(50)/MIC(90) of 32/64 mg/L. Erythromycin resistance rose from 1.9% before 2000 to 4% after 2000 and was associated with a marked increased of MIC levels. Simultaneously, both the phenotype and genotype of macrolide resistance were modified as the M phenotypes (mef(A) genotype) were replaced by the cMLS(B) phenotypes (erm(B) genotype). CONCLUSION: This polyclonal spreading of cMLS(B) macrolide resistance has not been previously observed in South America and should stimulate further epidemiological surveillance in this part of the world.

Group A Streptococcus virulence and host factors in two toddlers with rheumatic fever following toxic shock syndrome.

BACKGROUND: Rheumatic fever (RF) classically occurs after group A Streptococcus (GAS) pharyngitis in children aged over 5 years in developing countries. The present report describes the bacterial and host determinants in non-related toddlers who developed RF diagnostic criteria after toxic shock syndrome (TSS). METHODS AND RESULTS: A 13-month-old boy and a 14-month-old girl presented GAS TSS. After several weeks, multiple subcutaneous nodules as well as migratory polyarthritis or monoarthritis developed in both children, fulfilling Jones criteria of RF. The relevance of the Jones criteria for very young children is, however, debatable and their use might lead to the unnecessary prescribing of secondary prophylaxis. A molecular analysis of both bacterial and host factors was carried out in an attempt to decipher the combination that could have led to such uncommon, but very similar presentations. The two GAS isolates belonged to the usual, although distinct, invasive emm-types 1 and 3. Both isolates carried a wide set of prophage-encoded virulence factors, with only the speG and speA superantigen-encoding genes in common. Both patients shared the HLA DQB1*0301 allele, which has been associated with susceptibility to GAS necrotizing fasciitis. CONCLUSIONS: Our study exemplifies the particularity of RF in young children and the complex role of superantigens and streptodornases in GAS-related pathologies.

Polyclonal dissemination of tetracycline resistance among Streptococcus pyogenes paediatric isolates from Brazil.

INTRODUCTION: Scarce data are available on Group A Streptococcus (GAS) antibiotic resistance in South America. METHODOLOGY: The antibiotic susceptibility patterns of GAS recovered from symptomatic children living in the central part of Brazil during a prospective epidemiological study were analyzed. RESULTS: No isolates were resistant to penicillin or macrolides.  Sixty-one percent of the isolates were highly resistant to tetracycline, of which 85% harboured the tetM resistance gene. Ninety-five percent of these tetracycline resistant isolates were also resistant to minocycline. Thirty different emm-types were associated with tetracycline resistance. Phylogenetic analysis indicates that tetracycline resistance arose independently in distantly related emm-types. CONCLUSIONS: A high level of GAS tetracycline resistance has been observed in the central part of Brazil due to the polyclonal dissemination of resistant emm-types.

Emerging fluoroquinolone-non-susceptible group A streptococci in two different paediatric populations.

Clonal emergence of group A streptococci (GAS) with reduced susceptibility to fluoroquinolones (FQs) has been increasingly reported. Non-susceptibility is associated with various point mutations in the target-encoding genes and has only been described in a few emm types. We used a well-characterised GAS clinical paediatric collection from Brussels (Belgium) and Brasília (Brazil) to analyse the molecular basis of FQ non-susceptibility. GAS strains were tested for ciprofloxacin susceptibility and were screened for mutations in DNA gyrase- and topoisomerase IV-encoding genes. Genetic relationships between the different emm types were assessed by phylogenetic analysis of the whole surface-exposed part of the M protein. A high proportion (22.5%) of ciprofloxacin-non-susceptible isolates (minimal inhibitory concentration > or = 2mg/L) was found among the Belgian strains. They belonged mostly to emm type 6 (87%). In Brazil, 6% of the isolates, belonging to seven distantly related emm types, were non-susceptible. Our phylogenetic analysis showed that non-susceptibility may arise in various genetic backgrounds. Sequence comparison of the quinolone resistance-determining regions (QRDRs) of the ParC- and ParE-encoding genes from susceptible and non-susceptible isolates revealed that most of the mutations were found in both classes of isolates, indicating an emm type-linked polymorphism. In conclusion, we observed a clonal spreading of non-susceptible emm type 6 GAS strains in Brussels and a polyclonal distribution of non-susceptible isolates in Brazil. All the Brazilian and Belgian emm type 6 strains displayed a S79A/F mutation in parC that convincingly explains the non-susceptible phenotype.

Differences between Belgian and Brazilian group A Streptococcus epidemiologic landscape.

BACKGROUND: Group A Streptococcus (GAS) clinical and molecular epidemiology varies with location and time. These differences are not or are poorly understood. METHODS AND FINDINGS: We prospectively studied the epidemiology of GAS infections among children in outpatient hospital clinics in Brussels (Belgium) and Brasília (Brazil). Clinical questionnaires were filled out and microbiological sampling was performed. GAS isolates were emm-typed according to the Center for Disease Control protocol. emm pattern was predicted for each isolate. 334 GAS isolates were recovered from 706 children. Skin infections were frequent in Brasília (48% of the GAS infections), whereas pharyngitis were predominant (88%) in Brussels. The mean age of children with GAS pharyngitis in Brussels was lower than in Brasília (65/92 months, p<0.001). emm-typing revealed striking differences between Brazilian and Belgian GAS isolates. While 20 distinct emm-types were identified among 200 Belgian isolates, 48 were found among 128 Brazilian isolates. Belgian isolates belong mainly to emm pattern A-C (55%) and E (42.5%) while emm pattern E (51.5%) and D (36%) were predominant in Brasília. In Brasília, emm pattern D isolates were recovered from 18.5% of the pharyngitis, although this emm pattern is supposed to have a skin tropism. By contrast, A-C pattern isolates were infrequently recovered in a region where rheumatic fever is still highly prevalent. CONCLUSIONS: Epidemiologic features of GAS from a pediatric population were very different in an industrialised country and a low incomes region, not only in term of clinical presentation, but also in terms of genetic diversity and distribution of emm patterns. These differences should be taken into account for designing treatment guidelines and vaccine strategies.

Pharyngitis in low-resources settings: a pragmatic clinical approach to reduce unnecessary antibiotic use.

OBJECTIVE: Existing scoring systems for the diagnosis of group A streptococcus pharyngitis are insensitive or inapplicable in low-resources settings. Bacterial cultures and rapid tests can allow for antibiotic prescription abstention in high-income regions. These techniques are not feasible in many low-resources settings, and antibiotics often are prescribed for any pharyngitis episode. However, judicious antibiotics prescription in the community also is of concern in low-income countries. The objective of this study was to develop a clinical decision rule that allows for the reduction of empirical antibiotic therapy for children with pharyngitis in low-resources settings by identifying non-group A streptococcus pharyngitis. PATIENTS AND METHODS: We prospectively included children with pharyngitis in 3 public hospitals of Brazil during 9 months in 2004. We filled out clinical questionnaires and performed throat swabs. Bilateral chi2 (2-tailed test) and multivariate analysis were used to determine score categories. The outcome measures were sensitivity, specificity, positive likelihood ratio, and posttest probability of non-group A streptococcus infection with the clinical approach as compared with throat culture. RESULTS: A total of 163 of the 220 children had non-group A streptococcus pharyngitis (negative culture). We established a 3-questions decision rule (age and viral and bacterial signs) with 3 possible answers. The use of this score would prevent 41% to 55% of unnecessary antimicrobial prescriptions. The specificity of the score for non-group A streptococcus pharyngitis was >84%. CONCLUSION: Such a clinical decision rule could be helpful to reduce significantly unnecessary antibiotic prescriptions for pharyngitis in children in low-resources settings.

Randomize groups, not individuals: a strategy for improving early childhood programs.

Until the past few years, our nation's approach to designing federal programs for preschool-age children lacked coherence and paid little attention to what had worked (and not worked) in the past. In this article, the authors propose that credible information useful for designing effective programs will require the ongoing, systematic development and evaluation of alternative approaches for the improvement of large-scale early childhood programs. The research should place greater reliance on experiments in which existing groups of individuals, such as intact classes or preschool agencies, are randomly assigned to implement competing early education programs or program components. Randomizing groups, rather than individual children, changes the research question from "What works?" to "What works better?" yielding more useful information than is currently available about which preschool approaches ought to be strongly embedded in our nation's social policy.

Effects of a family literacy program on low-literate children and their parents: findings from an evaluation of the Even Start family literacy program.

A randomized experiment was conducted to test the effectiveness of Even Start, a federally supported family literacy program providing early childhood education, adult education, parenting education, and joint parent-child literacy activities to children and parents from low-literate families. The evaluation of 18 Even Start projects followed 463 families for 2 years and found no statistically significant or educationally important impacts on Even Start families when they were compared with control families on child literacy outcomes, parent literacy outcomes, or parent-child interactions. The study concludes that Even Start projects were able to properly implement family literacy programs, and the observed lack of effectiveness is attributed to a combination of 2 factors: (a) a lack of full participation on the part of families and (b) instructional services that may be ineffective because of the curriculum content or the instructional approach.

Red cell morphology and the peripheral blood film.

Red cell morphology as determined by microscopic examination of a stained blood film is an old and traditional approach to the evaluation of an anemic patient. The examination of a well-made and well-stained peripheral blood film remains an important and vital tool in the evaluation of the anemic patient and provides direction for the subsequent laboratory evaluation of the patient's anemia. This article provides a review of the important red cell changes necessary for evaluation in determining the cause of anemia.